(i) Field of the Invention
The present invention relates to an improved method to obtain male-sterile plants using foreign male-sterility genes that comprise plant promoters that direct expression of a male-sterility DNA in stamen cells, and to plants obtained by the method.
(ii) Description of Related Art
In many, if not most plant species, the development of hybrid cultivars is highly desired because of their generally increased productivity due to heterosis: the superiority of performance of hybrid individuals compared with their parents (see e.g. Fehr, 1987, Principles of cultivar development, Volume 1: Theory and Technique, MacMillan Publishing Company, New York; Allard, 1960, Principles of Plant Breeding, John Wiley and Sons, Inc.).
The development of hybrid cultivars of various plant species depends upon the capability to achieve almost complete cross-pollination between parents. This is most simply achieved by rendering one of the parent lines male sterile (i.e., bringing them in a condition so that pollen is absent or nonfunctional) either manually, by removing the anthers, or genetically by using, in the one parent, cytoplasmic or nuclear genes that prevent anther and/or pollen development (for a review of the genetics of male sterility in plants see Kaul, 1988, `Male Sterility in Higher Plants`, Springer Verlag).
For hybrid plants where the seed is the harvested product (e.g., corn, oilseed rape) it is in most cases also necessary to ensure that fertility of the hybrid plants is fully restored. In systems in which the male sterility is under genetic control this requires the existence and use of genes that can restore male fertility. The development of hybrid cultivars is mainly dependent on the availability of suitable and effective sterility and restorer genes.
Endogenous nuclear loci are known for most plant species that may contain genotypes which effect male sterility, and generally, such loci need to be homozygous for particular recessive alleles in order to result in a male-sterile phenotype. The presence of a dominant `male fertile` allele at such loci results in male fertility.
Recently it has been shown that male sterility can be induced in a plant by providing the genome of the plant with a chimeric male-sterility gene comprising a DNA sequence (or male-sterility DNA) coding, for example, for a cytotoxic product (such as an RNase) and under the control of a promoter which is predominantly active in selected tissue of the male reproductive organs. In this regard stamen-specific promoters, such as the promoter of the TA29 gene of Nicotiana tabacum, have been shown to be particularly useful for this purpose (Mariani et al., 1990, Nature 347:737, European patent publication ("EP") 0,344,029). By providing the nuclear genome of the plant with such a male-sterility gene, an artificial male-sterility is locus is created containing the artificial male-sterility genotype that results in a male-sterile plant. Various stamen-specific promoters have been described (see e.g., WO 92/13956, WO 92/13957).
In addition it has been shown that male fertility can be restored to the plant with a chimeric fertility-restorer gene comprising another DNA sequence (or fertility-restorer DNA) that codes, for example, for a protein that inhibits the activity of the cytotoxic product or otherwise prevents the cytotoxic product to be active in the plant cells (EP 0,412,911). For example the barnase gene of Bacillus amyloliquefaciens codes for an RNase, the barnase, which can be inhibited by a protein, the barstar, that is encoded by the barstar gene of B. amyloliquefaciens. The barnase gene can be used for the construction of a sterility gene while the barstar gene can be used for the construction of a fertility-restorer gene. Experiments in different plant species, e.g., oilseed rape, have shown that a chimeric barstar gene can fully restore the male fertility of male sterile lines in which the male sterility was due to the presence of a chimeric barnase gene (EP 0,412,911, Mariani et al., 1991, Proceedings of the CCIRC Rapeseed Congress, Jul. 9-11, 1991, Saskatoon, Saskatchewan, Canada; Mariani et al., 1992, Nature 357:384). By coupling a marker gene, such as a dominant herbicide resistance gene (for example the bar gene coding for phosphinothricin acetyl transferase (PAT) that converts the herbicidal phosphinothricin to a non-toxic compound [De Block et al., 1987, EMBO J. 6:2513]), to the chimeric male-sterility and/or fertility-restorer gene, breeding systems can be implemented e.g. to select for uniform populations of male sterile plants (EP 0,344,029; EP 0,412,911).
Barnase is an extracellular ribonuclease produced by Bacillus amyloliquefaciens. Barstar is an inhibitor of barnase that is produced intracellularly by the same bacterium to protect it from the toxic effects of the intracellular barnase activity (Hartley, 1989, TIBS, 14:450-454). Initial attempts to clone the barnase gene in E.coli and B.subtilis under control of its own or another bacterial promoter were unsuccesful as the produced barnase proved to be toxic to the host cells. When the bamase gene was reconstructed from previously cloned parts on the same plasmid as the barstar gene, the lethal effects of barnase expression were suppressed (Hartley, 1988, J. Mol. Biol. 202:913-915).
Whenever barnase is cloned in a bacterial host cell, such as E.coli, it may be useful to have the barstar gene, under control of its native or another bacterial promoter, present in the host cell to prevent possible harmful effects of undesired barnase expression. Paul et al, 1992, Plant Mol. Biol. 19:611-622 for instance, constructed a chimeric barnase gene under control of a tapetum specific promoter of the A9 gene of Arabidopsis. Plasmids pWP127 and pWP128 contain a DNA fragment encoding barstar and the mature barnase cloned between the 1437 bp A9 promoter fragment and a CaMV polyadenylation sequence. The promoter and coding sequence of barstar were included on these plasmids since mature barnase could not be cloned in its absence in E.coli.
As indicated above barnase DNA has been used to induce male-sterility in plants. However, other uses of barnase have also been described. WO 92/21757 describes inter alia a plant transformed with a nematode-induced chimaeric gene comprising the following operably linked DNA sequences:
a nematode-induced promoter that is suitable to direct transcription of a foreign DNA substantially selectively in specific root cells, preferably in the cells of fixed-feeding sites of the plant; and, PA1 a first foreign DNA that encodes barnase; PA1 a second promoter, such as a nematode-repressed promoter, which can direct transcription of a second foreign DNA in cells of the plant where the first foreign DNA is expressed, preferably substantially selectively in cells other than the specific root cells, preferably in cells other than the fixed feeding site cells, of the plant, and, PA1 a second foreign DNA that encodes barstar. PA1 a fungus-responsive promoter that is suitable to direct transcription of a foreign DNA substantially selectively in cells of a plant surrounding, preferably immediately surrounding, a site of infection of the plant by a fungus; and, PA1 a first foreign DNA that encodes barnase; PA1 a second promoter, such as a constitutive promoter (e.g., 35S), which can direct transcription of a second foreign DNA in cells of the plant other than those surrounding, preferably in at least cells of the plant other than those immediately surrounding, said fungus infection site; and, PA1 a second foreign DNA that encodes barstar. PA1 a male-sterility gene comprising: PA1 a coregulating gene comprising: PA1 transforming the nuclear genome of plant cells with a foreign DNA comprising a male-sterility gene comprising: PA1 regenerating plants transformed with said foreign DNA from said transformed cells, which method is characterized by including in said foreign DNA a coregulating gene comprising a coregulating DNA encoding a coregulating RNA, protein or polypeptide, preferably barstar, which is capable, when produced in plant cells wherein said sterility RNA, protein or polypeptide is produced, of sufficiently preventing the activity of said sterility RNA, protein or polypeptide, said coregulating DNA preferably being under the control of a promoter including: PA1 t1) a marker DNA encoding a marker RNA, protein or polypeptide which, when present at least in a specific tissue or specific cells of the plant, renders the plant easily separable from other plants which do not contain the marker RNA, protein or polypeptide encoded by the marker DNA at least in the specific tissue or specific is cells, and, PA1 t2) a marker promoter capable of directing expression of the marker DNA at least in the specific tissue or specific cells: the marker DNA being in the same transcriptional unit as, and under the control of, the marker promoter. PA1 low-level expression of the male-sterility gene in some transformed plant cells in tissue culture, including regeneration prior to normal plant development. Indeed such tissue culture cells have a physiology and metabolism and patterns of gene regulation which may be different from that of any differentiated cell in a plant or seed. Since the sterility promoter is generally selected on the basis of its natural activity in the plant or seed, position effects are perhaps expected to be more pronounced to activate the promoter in tissue culture cells. When direct gene transfer is used an additional phenomenon may occur. Indeed in such transformation method a large amount of DNA is delivered to any recipient cell. If gene repression should be an active process, which requires for instance DNA methylation or repressor protein binding, the repression mechanism may become temporarily overloaded, and the delivered DNA may be expressed for a short period of time. It can be seen that the coregulating gene can thus increase the general transformation efficiency. PA1 low-level expression of the sterility DNA (e.g., the barnase DNA) in specific non-stamen cells of the primary transformants and/or particularly the progeny plants obtained thereof. Such low level expression can be due to several factors many of which are largely unknown:
and which also contains a restorer chimaeric gene, preferably in the same genetic locus as the nematode-induced chimaeric gene, comprising the following operably linked DNA sequences:
WO 93/19188 describes inter alia a plant transformed with a fungus-responsive chimaeric gene comprising the following operably linked DNA sequences:
and which also contains a restorer chimaeric gene, preferably in the same genetic locus as the fungus-responsive chimaeric gene, comprising the following operably linked DNA sequences:
A foreign DNA, when introduced in the plant genome appears to integrate randomly in the plant genome. Examination of independently transformed plants has shown a high degree of variability (up to 100-fold) in the expression level of the introduced gene. Several studies have shown no correlation between this "between-transformant variability" and the copy number of the introduced DNA at a given locus. It has been suggested that some of the variability in expression of introduced genes in transgenic plants is a consequence of "position effects" caused by influences of adjacent plant genomic DNA. Other factors that could contribute to the variability in expression are physiological variability of the plant material, differences in the number of independent T-DNA loci in different transformants or the inhibitory effects of certain T-DNA structures on gene expression. Between-transformant variability in expression has been observed for the majority of introduced genes in transgenic plants. The variability in expression of many introduced genes in independent transgenic plants necessitates large numbers of transgenic plants to be assayed to accurately quantitate the expression of the gene. It would be of great importance if the amount of between-transformant variability could be reduced (Dean et al, 1988, NAR 16:9267-9283).